| 1. |
- Baladi, Tom, 1991, et al.
(författare)
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Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery
- 2021
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 143:14, s. 5413-5424
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Tidskriftsartikel (refereegranskat)abstract
- Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.
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| 2. |
- Börjesson, Karl, 1982, et al.
(författare)
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Membrane-Anchored DNA Assembly for Energy and Electron Transfer
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:8, s. 2831-2839
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Tidskriftsartikel (refereegranskat)abstract
- In this work we examine the trapping and conversion of visible light energy into chemical energy using a supramolecular assembly. The assembly consists of a light-absorbing antenna and a porphyrin redox center, which are covalently attached to two complementary 14-mer DNA strands, hybridized to form a double helix and anchored to a lipid membrane. The excitation energy Is finally trapped In the lipid phase of the membrane as a benzoquinone radical anion that could potentially be used In subsequent chemical reactions. In addition, In this model complex, the hydrophobic porphyrin moiety acts as an anchor into the liposome positioning the DNA construct on the lipid membrane surface. The results show the suitability of our system as a prototype for DNA-based light-harvesting devices, In which energy transfer from the aqueous phase to the interior of the lipid membrane Is followed by charge separation. © 2009 American Chemical Society.
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| 3. |
- Börjesson, Karl, 1982, et al.
(författare)
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Nucleic Acid Base Analog FRET-Pair Facilitating Detailed Structural Measurements in Nucleic Acid Containing Systems
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:12, s. 4288-4293
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Tidskriftsartikel (refereegranskat)abstract
- We present the first nucleobase analog fluorescence resonance energy transfer (FRET)-pair. The pair consists of tC O , 1,3-diaza-2- oxophenoxazine, as an energy donor and the newly developed tC nitro , 7-nitro-1,3-diaza-2-oxophenothiazine, as an energy acceptor. The FRET-pair successfully monitors distances covering up to more than one turn of the DNA duplex. Importantly, we show that the rigid stacking of the two base analogs, and consequently excellent control of their exact positions and orientations, results in a high control of the orientation factor and hence very distinct FRET changes as the number of bases separating tC O and tCnitro is varied. A set of DNA strands containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately distinguish distance- from orientation-changes using FRET. In combination with the good nucleobase analog properties, this points toward detailed studies of the inherent dynamics of nucleic acid structures. Moreover, the placement of FRET-pair chromophores inside the base stack will be a great advantage in studies where other (biomacro)molecules interact with the nucleic acid. Lastly, our study gives possibly the first truly solid experimental support to the dependence of energy transfer efficiency on orientation of involved transition dipoles as predicted by the Forster theory. © 2009 American Chemical Society.
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| 4. |
- Kumar, R., et al.
(författare)
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Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry
- 2007
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 129:21, s. 6859-6864
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Tidskriftsartikel (refereegranskat)abstract
- The copper-catalyzed azide-alkyne cycloaddition reaction has been used for the template-mediated chemical ligation of two oligonucleotide strands, one with a 5'-alkyne and the other with a 3'-azide, to produce a DNA strand with an unnatural backbone at the ligation point. A template-free click-ligation reaction has been used for the intramolecular circularization of a single stranded oligonucleotide which was used as a template for the synthesis of a covalently closed DNA catenane.
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| 5. |
- Olofsson, Johan, 1974, et al.
(författare)
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Effects of methyl substitution on radiative and solvent quenching rate constants of [Ru(phen)(2)dppz](2+) in polyol solvents and bound to DNA
- 2004
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 126:47, s. 15458-15465
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Tidskriftsartikel (refereegranskat)abstract
- Methyl substituents on the distant benzene ring of the dppz ligand in the "light switch" complex [Ru(phen)(2)dppz](2+) have profound effects on the photophysics of the complexes in water as well as in the polyol solvents ethylene glycol, glycerol, and 1,2- and 1,3-propanediol. Whereas 11,12-dimethyl substitution decreases the rate of quenching by diminishing hydrogen bonding by solvent, the 10-methyl substituent in addition also decreases both the radiative and the nonradiative rate constant for decay to the ground state of the non-hydrogen-bonded excited state species. For both the 10-methyl and the 11,12-dimethyl derivatives, the effect of methyl substitution on the equilibrium of solvent hydrogen bonding to the excited state is due to changes in the entropy terms, rather than in the enthalpy; indicating that the effect is a steric perturbation of the solvent cage around the molecule. When intercalated into DNA, the effects of methyl substitution is smaller than those in polyol solvent or water, suggesting that the water molecules that quench the excited state by hydrogen bonding to the phenazine aza nitrogens mainly access them from the same groove as in which the Ru(II) ion resides. Since the Delta-enantiomer of [Ru(phen)(2)10-methyl-dppz](2+) has an absolute quantum yield of up to 0.23 when bound to DNA, a value 7000 times higher than in pure water solution, it is promising as a new luminescent DNA probe.
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| 6. |
- Sandberg Wranne, Moa, 1986, et al.
(författare)
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Toward Complete Sequence Flexibility of Nucleic Acid Base Analogue FRET
- 2017
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 139:27, s. 9271-9280
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Tidskriftsartikel (refereegranskat)abstract
- Forster resonance energy transfer (FRET) using fluorescent base analogues is a powerful means of obtaining high-resolution nucleic acid structure and dynamics information that favorably complements techniques such as NMR and X-ray crystallography. Here, we expand the base base FRET repertoire with an adenine analogue FRET-pair. Phosphoramidite-protected quadracyclic 2'-deoxyadenosine analogues qAN1 (donor) and qA(nitro) (acceptor) were synthesized and incorporated into DNA by a genetic, reliable, and high-yielding route, and both constitute excellent adenine analogues. The donor, qAN1, has quantum yields reaching 21% and 11% in single- and double-strands, respectively. To the best of our knowledge, this results in the highest average brightness of an adenine analogue inside DNA. Its potent emissive features overlap well with the absorption of qA(nitro), and thus enable accurate FRET-measurements over more than one turn of B-DNA. As we have shown previously for our cytosine analogue FRET-pair, FRET between qAN1 and qA(nitro) positioned at different base separations inside DNA results in efficiencies that are highly dependent on both distance and orientation. This facilitates significantly enhanced resolution in FRET structure determinations, demonstrated here in a study of conformational changes of DNA upon binding of the minor groove binder netropsin. Finally, we note that the donor and acceptor, of our cytosine FRET-pair, tC degrees and tC(nitro), can be conveniently combined with the acceptor and donor of our current adenine pair, respectively. Consequently, our base analogues can now measure base base FRET between 3 of the 10 possible base combinations and, through base-complementarity, between all sequence positions in a duplex.
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| 7. |
- Westerlund, Fredrik, 1978, et al.
(författare)
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Micelle-sequestered dissociation of cationic DNA-intercalated drugs: Unexpected surfactant-induced rate enhancement
- 2003
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 125:13, s. 3773-3779
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Tidskriftsartikel (refereegranskat)abstract
- Detergent sequestration using micelles as a hydrophobic sink for dissociated drug molecules is an established technique for determination of dissociation rates. The anionic surfactant molecules are generally assumed not to interact with the anionic DNA and thereby not to affect the rate of dissociation. By contrast, we here demonstrate that the surfactant molecules sodium dodecyl sulfate (SDS), sodium decyl sulfate, and sodium octyl sulfate all induce substantial rate enhancements of the dissociation of intercalators from DNA. Four different cationic DNA intercalators are studied with respect to surfactant-induced dissociation. Except for the smallest intercalator, ethidium, the dissociation rate constants increase monotonically with surfactant concentration both below cmc and (more strongly) above cmc, much more than expected from electrostatic effects of increased counterion concentration. The rate enhancement, most pronounced for the bulky, multicationic, hydrophobic DNA ligands in this study, indicates a reduction of the activation energy for the ligand to pass out from a deeply penetrating intercalation site of DNA. The discovery that surfactants enhance the rate of dissociation of cationic DNA-intercalators implies that rate constants previously determined by micelle-sequestered dissociation may have been overestimated. As an alternative, more reliable method, we suggest instead the addition of excess of dummy DNA as an absorbent for dissociated ligand.
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| 9. |
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